Production of L-isoleucine by means of recombinant microorganisms with deregulated threonine dehydratase

Abstract

PCT No. PCT/DE95/00017 Sec. 371 Date Mar. 20, 1997 Sec. 102(e) Date Mar. 20, 1997 PCT Filed Jan. 9, 1995 PCT Pub. No. WO95/19442 PCT Pub. Date Jul. 20, 1995The invention relates to processes for the microbial production of L-isoleucine. To this end, in a gene in vitro of a threonine dehydratse, one or more bases in the gene region coding the enzyme's allosteric domains are exchanged in such a way that at least one amino acid in the amino acid sequence of the allosteric domains of the enzyme is replaced by another so that the enzyme is no longer inhibited by L-isoleucine feedback. Furthermore, concrete amino acid exchanges in the amino acid sequence of the enzyme are effected in a gene in vitro of a threonin dehydratase of Corynebacterium glutamicum by base exchange both outside and inside and outside the gene region coding the allosteric domains of the enzyme si that, after the transformation of such mutated threonine dehydratase genes into a threonine or L-isoleucine-producing host cell, the latter repeatedly forms L-isoleucine.

We claim: 
     
       1. A method for the microbial production of L-isoleucine, comprising (A) recombinantly preparing a microorganism having a mutated threonine dehydratase gene by replacing at least one base in the region of the gene that encodes an allosteric domain of the threonine dehydratase, wherein said replacing at least one base results in deregulation of threonine dehydratase from L-isoleucine feedback inhibition, thereby producing a recombinant microorganism, and   (B) culturing the recombinant microorganism to produce L-isoleucine.   
     
     
       2. The method of claim 1, wherein the microorganism is Corynebacterium glutamicum. 
     
     
       3. The method of claim 1, wherein the recombinant microorganism is obtained by growing the microorganism on a solid medium which contains L-isoleucine and L-valine, and then selecting colonies with an altered morphology. 
     
     
       4. The method of claim 1, wherein said mutated threonine dehydratase gene is obtained from Escherichia coli. 
     
     
       5. The method of claim 1, wherein said mutated threonine dehydratase gene is obtained from Corynebacterium glutamicum. 
     
     
       6. The method of claim 1, wherein said replacing is selected from the group consisting of (i) replacing alanine in position 257 of SEQ ID NO: 8 with glycine and (ii) replacing methionine in position 199 of SEQ ID NO: 10 with valine. 
     
     
       7. The method of claim 1, wherein said replacing includes replacing, in SEQ ID NO:10, (a) histidine in position 278 with arginine and (b) leucine in position 351 with serine. 
     
     
       8. The method of claim 3, wherein acetohydroxy acid synthase activity of the microorganism has been inhibited by the L-valine contained in the solid medium. 
     
     
       9. The method of claim 3, wherein the medium comprises a substance for inducing the threonine dehydratase gene. 
     
     
       10. The method of claim 9, wherein said substance is isopropyl-β-D-thiogalactopyranoside. 
     
     
       11. An isolated DNA that encodes a threonine dehydratase that is insensitive to L-isoleucine feedback, said DNA prepared by replacing at least one base in the region of the DNA that encodes an allosteric domain of the threonine-dehydratase. 
     
     
       12. The DNA of claim 11, comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5. 
     
     
       13. The DNA of claim 11, comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:9, and SEQ ID NO:11. 
     
     
       14. A cell comprising the DNA of claim 11. 
     
     
       15. A vector comprising DNA that encodes a threonine dehydratase that is insensitive to L-isoleucine feedback, said DNA prepared by replacing at least one base in the region of the DNA that encodes an allosteric domain of the threonine-dehydratase. 
     
     
       16. A vector according to claim 15, further comprising a promoter, inducible by isopropyl-β-D-thiogalactopyranoside, that is operably associated with said DNA. 
     
     
       17. A cell comprising a vector according to claim 15. 
     
     
       18. A cell comprising a vector according to claim 16. 
     
     
       19. The cell as claimed in claim 18, wherein said cell exhibits acetohydroxy acid synthase activity which can be inhibited by L-valine. 
     
     
       20. The cell as claimed in claim 18, which is an Escherichia coli cell. 
     
     
       21. The cell as claimed in claim 18, which is a Corynebacterium glutamicum cell. 
     
     
       22. The cell as claimed in claim 18, wherein said cell does not synthesize threonine dehydratase that is sensitive to L-isoleucine feedback.